Why is camp a second messenger

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Single On Purpose: Redefine Everything. Find Yourself First. John Kim. Permission to Dream Chris Gardner. Gundry, MD. Shree Shree. The recognition process between intracellular second messengers and extracellular receptors gives rise to a series of biochemical reactions that result in several physiological effects.

Schematic diagram of second messenger signaling pathways. Second messengers convert and amplify extracellular signals by activating protein kinases that serve physiological roles or by acting on intracellular ligand-gated channels to alter the membrane potential. The degradation of these second messengers leads to signal termination. This review focuses primarily on reviewing cAMP, an important second messenger, and the associated cell signal transduction pathway.

Signal response factors associated with cAMP are discussed below and the current understanding of the cAMP signaling pathway is presented in Fig. Subsequently, specific proteins are phosphorylated by PKA 2 to evoke cellular reactions.

The phosphorylation of the cAMP response-element binding-protein CREB , a transcription factor, is important in the regulation of gene transcription 3. Extracellular signals activate the transcription of a variety of target genes via alterations in CREB phosphorylation, thereby, resulting in multiple physiological functions 4. Second messenger pathways are associated with numerous conditions and diseases, including inflammation 6 , 7 , cancer 8 , 9 , myocardial atrophy 2 , asynodia 10 and depression All of the conditions and diseases mentioned above involve the cAMP signaling pathway and its branch pathway.

Due to the importance and varied functions of the cAMP signaling pathway, Gloerich and Bos 12 and Nakajima et al 13 studied the underlying mechanisms in detail. The present review discusses the methods used to detect the cAMP signaling pathway, as well as the diseases associated with the pathway.

Research on cAMP signaling pathways requires the detection of the signaling system at various levels, including each target factor. With functions including the regulation of neurotransmitter synthesis 19 , regulation of membrane protein activity, participation in ganglion synaptic transmission 20 and regulation of transcription factors in eukaryotic cells 21 , cAMP may be involved in the prevention and treatment of various diseases.

Therefore, detecting the level of cAMP is important in the investigation of medically relevant signal transduction pathways. An immunochemical assay is a fast and effective method for detecting cAMP in the field of biomedical research. An RIA is a radionuclide-labeled immune analysis method. The basic principle of an RIA is a competitive binding reaction between a radioisotope-labeled antigen and an unlabeled antigen for a specific antibody. An RIA is a method that employs a competitive inhibition reaction and is characterized by high sensitivity, strong specificity and low cost.

An RIA is convenient for the early detection of biological samples, however there are concerns with this assay regarding experimental safety and environmental protection. This method is based on an immuno-competitive binding technique. This method depends on specific adsorption and the combination of the antibody and antigen. The cAMP in the sample or standard competes with a horseradish peroxidase HRP -labeled cAMP conjugate for binding sites on the anti-cAMP antibodies, and the results are measured with a multifunctional microplate reader to calculate the antibody or antigen concentration.

To improve the detection sensitivity, numerous commercial kits suggest pretreating the samples using acetylation. Fluorescent and chemiluminescent substrates 25 , which are able to greatly improve the sensitivity of detection, were subsequently developed. Shelton, CT, USA and is another alternative approach for determining cAMP levels 26 , is a homogeneous time-resolved fluorescence resonance energy transfer method.

The samples are appropriately prepared for time-resolved fluorescence measurements according to the manufacturer's instructions. Additionally, there are several other detection technologies, such as the scintillation proximity assay 27 and the high performance liquid chromatography-mass spectrometry HPLC-MS analysis technique.

By catalyzing phosphorylation in response to hormonal stimulation, PKA is the primary mediator of cAMP function and a key regulatory enzyme in pivotal cellular processes, such as DNA replication 28 , 29 , cell growth and metabolism 30 , cell division and rearrangement of the actin cytoskeleton 31 , Due to the fact that PKA is a type of protein kinase, the methods used in PKA research are divided into three groups: The detection of kinase activity, mRNA expression levels and protein expression levels.

The RT-PCR products were then separated by agarose gel electrophoresis, and the results analyzed with a gel image-analysis system. Western blot analysis is used to detect the expression of PKA proteins. In western blot analysis, polyacrylamide gel electrophoresis PAGE is used to separate proteins, and immunochemical staining or autoradiography is then used to detect the electrophoretically separated protein expressed by a specific gene.

Commercially available kits for the rapid detection of protein kinases have been used for many years. According to the characteristics of a phosphate kinase, detection technology was developed utilizing radioactive phosphorus 32 32 P as a marker. Although this method is effective, the large quantity of 32 P used makes this assay inconvenient and potentially hazardous. However, a non-radioactive protein kinase assay 38 for PKA is now available. This system is based on the high affinity binding of biotin to streptavidin.

In addition, a fluorescent peptide substrate is used. This method was designed to be rapid, sensitive and safe. Due to experimental safety and environmental protection considerations, researchers have been trying to develop increased numbers of non-radioactive detection technologies to detect the activity of PKA.

HPLC-MS, which uses liquid chromatography as the separation system and mass spectrometry as the detection system, has been suggested to be effective for measuring PKA activity. Samples are separated by mass in a packed column under high-pressure flow. The samples are then ionized, and the mass analyzer separates the ion fragments in accordance with mass number.

The mass spectra are created by the detector. Fujikawa et al 39 and Kanno et al 40 used a reversed-phase HPLC system in which phosphorylated and non-phosphorylated peptides were detected at an absorbance of nm.

This method combined the high separation capability of chromatography with the high selectivity and high sensitivity of mass spectrometry; therefore, HPLC-MS has the advantages of rapid analysis and amenability to automation.

CREB is a nuclear transcription factor. Non-phosphorylated CREB is predominantly located in the nucleus. PKA that is activated by cAMP translocates to the nucleus and activates CREB through the phosphorylation of the amino terminal kinase inducible domain, in turn regulating target gene transcription 41 — The N-terminus is the transcription activation site, which contains multiple phosphorylation sites, including serine residue Ser , Ser and Ser, which can be phosphorylated by a variety of protein kinases.

Ser serves an important role in the transcriptional activity of CREB 44 — The phosphorylation of these sites is associated with downstream protein expression and function.

Therefore, the detection of these phosphorylated sites is crucial to the study of signal transduction pathways. A variety of methods have been developed to detect CREB.

The most widely used is western blot analysis and transfection together with the luciferase assay. In western blotting method, the protein samples are separated by PAGE; the proteins are then transferred to membranes and subsequently probed with a specific antibody. Due to the importance of CREB and its phosphorylation, it has become a focus in research for targets of novel drug research and development.

The ELISA method, which is suitable for high throughput screening, is well established and is widely used in drug discovery. Therefore, the cell-based ELISA method based on the double fluorescent labeling technique has been widely used previously. In this method, an immobilized capture antibody specific for CREB binds to phosphorylated and unphosphorylated proteins. Subsequent to washing the unbound antibodies away, a biotinylated detection antibody that recognizes p-CREB Ser is used to detect only the phosphorylated protein, utilizing a standard streptavidin-HRP format.

The traditional detection method is an electrophoretic mobility shift assay EMSA. However, due to safety concerns regarding the use of radioactive isotopes, radioactive labeling has been replaced by non-radioactive visualization techniques. The luciferase reporter gene assay 54 involves the transfection of the reporter gene plasmid CREB-Luc into cells. The cells undergo appropriate stimulation and are then lysed, followed by treatment to detect luciferase activity.

This method can gauge the expression of a reporter gene easily and effectively. The construction of a reporter gene plasmid is accomplished by cloning the gene transcription regulatory elements upstream of, or at other appropriate locations relative to, the luciferase gene. Cells are transfected with the construct and luciferase activity is detected following treatment or proper stimulation.

The influences of different treatments on the targeted regulatory elements, or the differences prior to and subsequent to stimulation, are quantified using the luciferase activity level.

ChIP is an important method for investigating the interactions between specific proteins or modified forms of proteins and a genomic DNA region ChIP is based on the development of an in vivo analysis method. The basic principle is to selectively enrich a chromosomal fragment chromatin , which contains a specific antigen. An antibody that can identify a protein or modified protein is used to determine the relative abundance of the antigen at one or more locations in the genome.

A complex PDE gene organization and a great number of PDE splicing variants fine-tune cyclic nucleotide signals and make PDEs conducive to specificity in the signaling pathways 1. Inhibitors of PDE lead to the elevation of cAMP and cGMP levels, which in turn lead to multifarious cellular effects, including airway smooth muscle relaxation, inhibitory effects on cellular inflammation and immune responses The PDE4 inhibitors roflumilast 59 , 60 and cilomilast 61 have indicated the potential of the development of PDE inhibitors into novel drugs.

Previous studies have used a luminescence-based, high-throughput screening method in place of the enzyme kinetic method for measuring cyclic nucleotide PDE activity. As its name suggests, it hydrolyzes phospholipids — specifically phosphatidylinositol-4,5-bisphosphate PIP 2 which is found in the inner layer of the plasma membrane.

Hydrolysis of PIP 2 yields two products:. The calcium rise is needed for NF-AT the "nuclear factor of activated T cells" to turn on the appropriate genes in the nucleus. The remarkable ability of tacrolimus and cyclosporine to prevent graft rejection is due to their blocking this pathway. In fact, calcium ions are probably the most widely used intracellular messengers. However, its level in the cell can rise dramatically when channels in the plasma membrane open to allow it in from the extracellular fluid or from depots within the cell such as the endoplasmic reticulum and mitochondria.

Some factors at work:. John W. This content is distributed under a Creative Commons Attribution 3.